Yarrowia lipolytica as a Platform for Punicic Acid Production.

Punicic acid (PuA) is a polyunsaturated fatty acid with significant medical, biological, and nutraceutical properties. The primary source of punicic acid is the pomegranate seed oil obtained from fruits of trees that are mainly cultivated in subtropical and tropical climates. To establish sustainable production of PuA, various recombinant microorganisms and plants have been explored as platforms with limited efficiencies. In this study, the oleaginous yeast Yarrowia lipolytica was employed as a host for PuA production. First, growth and lipid accumulation of Y. lipolytica were evaluated in medium supplemented with pomegranate seed oil, resulting in the accumulation of lipids up to 31.2%, consisting of 22% PuA esterified in the fraction of glycerolipids. In addition, lipid-engineered Y. lipolytica strains, transformed with the bifunctional fatty acid conjugase/desaturase from Punica granatum (PgFADX), showed the ability to accumulate PuA de novo. PuA was detected in both polar and neutral lipid fractions, especially in phosphatidylcholine and triacylglycerols. Promoter optimization for PgFADX expression resulted in improved accumulation of PuA from 0.9 to 1.8 mg/g of dry cell weight. The best-producing strain expressing PgFADX under the control of a strong erythritol-inducible promoter produced 36.6 mg/L PuA. These results demonstrate that the yeast Y. lipolytica is a promising host for PuA production.

Heterologous Production of Calendic Acid Naturally Found in Calendula officinalis by Recombinant Fission Yeast.

Calendic acid (CA) is a conjugated fatty acid with anti-cancer properties that is widely present in seed oil of Calendula officinalis. Using the co-expression of C. officinalis fatty acid conjugases (CoFADX-1 or CoFADX-2) and Punica granatum fatty acid desaturase (PgFAD2), we metabolically engineered the synthesis of CA in the yeast Schizosaccharomyces pombe without the need for linoleic acid (LA) supplementation. The highest CA titer and achieved accumulation were 4.4 mg/L and 3.7 mg/g of DCW in PgFAD2 + CoFADX-2 recombinant strain cultivated at 16 °C for 72 h, respectively. Further analyses revealed the accumulation of CA in free fatty acids (FFA) and downregulation of the lcf1 gene encoding long-chain fatty acyl-CoA synthetase. The developed recombinant yeast system represents an important tool for the future identification of the essential components of the channeling machinery to produce CA as a high-value conjugated fatty acid at an industrial level.

Bioactivity and biotechnological production of punicic acid.

Punicic acid (PuA; 18: 3Δ 9cis,11trans,13cis ) is an unusual 18-carbon fatty acid bearing three conjugated double bonds. It has been shown to exhibit a myriad of beneficial bioactivities including anti-cancer, anti-diabetes, anti-obesity, antioxidant, and anti-inflammatory properties. Pomegranate (Punica granatum) seed oil contains approximately 80% PuA and is currently the major natural source of this remarkable fatty acid. While both PuA and pomegranate seed oil have been used as functional ingredients in foods and cosmetics for some time, their value in pharmaceutical/medical and industrial applications are presently under further exploration. Unfortunately, the availability of PuA is severely limited by the low yield and unstable supply of pomegranate seeds. In addition, efforts to produce PuA in transgenic crops have been limited by a relatively low content of PuA in the resulting seed oil. The production of PuA in engineered microorganisms with modern fermentation technology is therefore a promising and emerging method with the potential to resolve this predicament. In this paper, we provide a comprehensive review of this unusual fatty acid, covering topics ranging from its natural sources, biosynthesis, extraction and analysis, bioactivity, health benefits, and industrial applications, to recent efforts and future perspectives on the production of PuA in engineered plants and microorganisms.

Substrate preferences of long-chain acyl-CoA synthetase and diacylglycerol acyltransferase contribute to enrichment of flax seed oil with α-linolenic acid.

Seed oil from flax (Linum usitatissimum) is enriched in α-linolenic acid (ALA; 18:3Δ9cis,12cis,15cis ), but the biochemical processes underlying the enrichment of flax seed oil with this polyunsaturated fatty acid are not fully elucidated. Here, a potential process involving the catalytic actions of long-chain acyl-CoA synthetase (LACS) and diacylglycerol acyltransferase (DGAT) is proposed for ALA enrichment in triacylglycerol (TAG). LACS catalyzes the ATP-dependent activation of free fatty acid to form acyl-CoA, which in turn may serve as an acyl-donor in the DGAT-catalyzed reaction leading to TAG. To test this hypothesis, flax LACS and DGAT cDNAs were functionally expressed in Saccharomyces cerevisiae strains to probe their possible involvement in the enrichment of TAG with ALA. Among the identified flax LACSs, LuLACS8A exhibited significantly enhanced specificity for ALA over oleic acid (18:1Δ9cis ) or linoleic acid (18:2Δ9cis,12cis ). Enhanced α-linolenoyl-CoA specificity was also observed in the enzymatic assay of flax DGAT2 (LuDGAT2-3), which displayed ∼20 times increased preference toward α-linolenoyl-CoA over oleoyl-CoA. Moreover, when LuLACS8A and LuDGAT2-3 were co-expressed in yeast, both in vitro and in vivo experiments indicated that the ALA-containing TAG enrichment process was operative between LuLACS8A- and LuDGAT2-3-catalyzed reactions. Overall, the results support the hypothesis that the cooperation between the reactions catalyzed by LACS8 and DGAT2 may represent a route to enrich ALA production in the flax seed oil.